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1.
J Biol Chem ; 291(39): 20487-502, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27496950

RESUMO

Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.


Assuntos
Fenótipo de Síndrome de Antley-Bixler , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Fenótipo de Síndrome de Antley-Bixler/enzimologia , Fenótipo de Síndrome de Antley-Bixler/genética , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/metabolismo , Estabilidade Enzimática/genética , Humanos
2.
FEBS Lett ; 587(24): 3973-8, 2013 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-24211446

RESUMO

Neuronal nitric oxide synthase µ (nNOSµ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSµ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSµ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSµ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme-nitrosyl complex formation.


Assuntos
Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo I/fisiologia , Sequência de Aminoácidos/fisiologia , Animais , Calmodulina/química , Citocromos c/metabolismo , Transporte de Elétrons/fisiologia , Mononucleotídeo de Flavina/metabolismo , Heme/química , Heme/metabolismo , Isoenzimas/química , Isoenzimas/fisiologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo I/química , Ligação Proteica , Ratos
3.
PLoS One ; 8(9): e75638, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086598

RESUMO

NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Human patients with specific mutations in POR exhibit severe developmental malformations including disordered steroidogenesis, sexual ambiguities and various bone defects, similar to those seen in patients with Antley-Bixler syndrome (ABS). To probe the role of POR during bone development, we generated a conditional knockout mouse (CKO) by cross breeding Por (lox/lox) and Dermo1 Cre mice. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Differential staining of the CKO mice skull bases shows premature fusion of the sphenooccipital and basioccipital-exoccipital synchondroses. Class III malocclusion was noted in adult knockout mice with an unusual overgrowth of the lower incisors. Shorter long bones were observed along with a reduction in the bone volume fraction, measured by microCT, in the Por-deleted mice compared to age- and sex-matched littermate controls. Concerted up- or down-regulation of proteins in the FGF signaling pathway observed by immunohistochemistry in the tibia samples of CKO mice compared to wild type controls shows a decrease in the FGF signaling pathway. To our knowledge, this is the first report of a mouse model that recapitulates both skull and long bone defects upon Por deletion, offering an approach to study the sequelae of POR mutations. This unique model demonstrates that P450 metabolism in bone itself is potentially important for proper bone development, and that an apparent link exists between the POR and FGF signaling pathways, begging the question of how an oxidation-reduction flavoprotein affects developmental and cellular signaling processes.


Assuntos
Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , NADPH-Ferri-Hemoproteína Redutase/genética , Deleção de Sequência/genética , Células-Tronco/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/fisiopatologia , Animais , Fenótipo de Síndrome de Antley-Bixler/genética , Fenótipo de Síndrome de Antley-Bixler/metabolismo , Fenótipo de Síndrome de Antley-Bixler/fisiopatologia , Regulação para Baixo/genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Knockout , Mutação/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Células-Tronco/fisiologia , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismo , Regulação para Cima/genética
4.
Drug Metab Dispos ; 40(4): 754-60, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22252407

RESUMO

NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375-384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (k(cat) and K(m)) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained ∼10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (k(cat)/K(m)), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (k(cat)/K(m)) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.


Assuntos
Fenótipo de Síndrome de Antley-Bixler/enzimologia , Citocromo P-450 CYP3A/metabolismo , Variação Genética , NADPH-Ferri-Hemoproteína Redutase/genética , Fenótipo de Síndrome de Antley-Bixler/genética , Biotransformação , Catálise , Membrana Celular/enzimologia , Escherichia coli/genética , Fluoresceínas/metabolismo , Humanos , Técnicas In Vitro , Microssomos Hepáticos/enzimologia , Mutação , Plasmídeos , Especificidade por Substrato
5.
Proc Natl Acad Sci U S A ; 108(33): 13486-91, 2011 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-21808038

RESUMO

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.


Assuntos
Mutação de Sentido Incorreto , NADPH-Ferri-Hemoproteína Redutase/química , Dobramento de Proteína , Flavina-Adenina Dinucleotídeo , Humanos , Estrutura Molecular , NADPH-Ferri-Hemoproteína Redutase/deficiência , Polimorfismo Genético , Tripsina/metabolismo
6.
Arch Biochem Biophys ; 513(1): 42-50, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21741353

RESUMO

Genetic variations in POR, encoding NADPH-cytochrome P450 oxidoreductase (CYPOR), can diminish the function of numerous cytochromes P450, and also have the potential to block degradation of heme by heme oxygenase-1 (HO-1). Purified full-length human CYPOR, HO-1, and biliverdin reductase were reconstituted in lipid vesicles and assayed for NADPH-dependent conversion of heme to bilirubin. Naturally-occurring human CYPOR variants queried were: WT, A115V, Y181D, P228L, M263V, A287P, R457H, Y459H, and V492E. All CYPOR variants exhibited decreased bilirubin production relative to WT, with a lower apparent affinity of the CYPOR-HO-1 complex than WT. Addition of FMN or FAD partially restored the activities of Y181D, Y459H, and V492E. When mixed with WT CYPOR, only the Y181D CYPOR variant inhibited heme degradation by sequestering HO-1, whereas Y459H and V492E were unable to inhibit HO-1 activity suggesting that CYPOR variants might have differential binding affinities with redox partners. Titrating the CYPOR-HO-1 complex revealed that the optimal CYPOR:HO-1 ratio for activity was 1:2, lending evidence in support of productive HO-1 oligomerization, with higher ratios of CYPOR:HO-1 showing decreased activity. In conclusion, human POR mutations, shown to impact P450 activities, also result in varying degrees of diminished HO-1 activity, which may further complicate CYPOR deficiency.


Assuntos
Heme Oxigenase-1/química , Complexos Multienzimáticos/química , Mutação de Sentido Incorreto , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Multimerização Proteica , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Heme/química , Heme/genética , Heme/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo
7.
Biochem Biophys Res Commun ; 411(3): 490-5, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21726529

RESUMO

Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b(5) and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.


Assuntos
Conexina 43/genética , Junções Comunicantes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , NADPH-Ferri-Hemoproteína Redutase/fisiologia , Osteogênese/genética , Animais , Linhagem Celular Tumoral , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , NADPH-Ferri-Hemoproteína Redutase/genética
8.
Drug Metab Dispos ; 38(2): 332-40, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19884324

RESUMO

Patients with congenital adrenal hyperplasia, exhibiting combined CYP17 and CYP21 deficiency, were shown by Arlt et al. (2004) to harbor a 541T-->G mutation in exon 5 of POR (encoding NADPH-cytochrome P450 reductase, CYPOR), which resulted in a Y181D substitution that obliterated electron transfer capacity. Using bacterial expression models, we examined catalytic and physical properties of the human CYPOR Y181D variant. As purified, Y181D lacked flavin mononucleotide (FMN) and NADPH-cytochrome c reductase (NCR) activity but retained normal flavin adenine dinucleotide binding and NADPH utilization. Titration of the purified protein with FMN restored 64 of wild-type (WT) NCR activity in Y181D with an activation constant of approximately 2 microM. As determined by FMN fluorescence quenching, Y181D had K(d)(FMN) = 7.3 microM. Biplasmid coexpression of CYPOR and CYP1A2, at the physiological ratio of approximately 1:10 in the engineered MK_1A2_POR Escherichia coli strain, showed the compromised capacity of Y181D to support CYP1A2-catalyzed metabolism of the procarcinogens 2-aminoanthracene, 2-amino-3-methylimidazo(4,5-f)quinoline, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Isolated MK1A2_POR membranes confirmed FMN stimulation of Y181D NCR activity with a 1.6 microM activation constant. CYP1A2 ethoxyresorufin-O-dealkylase activity of the MK1A2_POR(Y181D) membranes, undetectable in the absence of added FMN, increased to 37% of MK1A2_POR(WT) membranes with a 1.2 microM FMN activation constant. Therefore, we conclude that compromised FMN binding is the specific molecular defect causing POR deficiency in patients with Y181D mutation and that this defect, in large part, can be overcome in vitro by FMN addition.


Assuntos
Mononucleotídeo de Flavina/metabolismo , NADPH-Ferri-Hemoproteína Redutase/deficiência , Substituição de Aminoácidos , Carcinógenos/metabolismo , Dicroísmo Circular , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Escherichia coli , Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Cinética , Membranas/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , NADH NADPH Oxirredutases/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Fluorescência , Espectrofotometria
9.
Chembiochem ; 10(4): 690-701, 2009 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-19222033

RESUMO

Nitric oxide synthases (NOSs) are unique flavohemoproteins with various roles in mammalian physiology. Constitutive NOS catalysis is initiated by fast hydride transfer from NADPH, followed by slower structural rearrangements. We used a photoactive nanotrigger (NT) to study the initial electron transfer to FAD in native neuronal NOS (nNOS) catalysis. Molecular modeling and fluorescence spectroscopy showed that selective NT binding to NADPH sites close to FAD is able to override Phe1395 regulation. Ultrafast injection of electrons into the protein electron pathway by NT photoactivation through the use of a femtosecond laser pulse is thus possible. We show that calmodulin, required for NO synthesis by constitutive NOS, strongly promotes intramolecular electron flow (6.2-fold stimulation) by a mechanism involving proton transfer to the reduced FAD(-) site. Site-directed mutagenesis using the S1176A and S1176T mutants of nNOS supports this hypothesis. The NT synchronized the initiation of flavoenzyme catalysis, leading to the formation of NO, as detected by EPR. This NT is thus promising for time-resolved X-ray and other cellular applications.


Assuntos
Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/biossíntese , Sítios de Ligação , Biocatálise , Calmodulina/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Flavina-Adenina Dinucleotídeo/metabolismo , Ligação de Hidrogênio , Cinética , Modelos Moleculares , NADP/metabolismo , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/genética , Processos Fotoquímicos , Mutação Puntual , Estrutura Terciária de Proteína , Espectrometria de Fluorescência
10.
Arch Biochem Biophys ; 475(2): 93-9, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18455494

RESUMO

Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR(null), POR(wt), POR(YH), and POR(VE), for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR(null), not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR(YH) and POR(VE) models than in POR(wt), indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.


Assuntos
Craniossinostoses/genética , Citocromo P-450 CYP1A2/genética , Flavina-Adenina Dinucleotídeo/química , Mutação , NADPH-Ferri-Hemoproteína Redutase/genética , Catálise , Formazans/metabolismo , Humanos , NADPH-Ferri-Hemoproteína Redutase/fisiologia , Oxirredução , Síndrome , Sais de Tetrazólio/metabolismo , Xenobióticos/metabolismo
11.
J Biol Chem ; 281(47): 35975-82, 2006 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-16998238

RESUMO

Numerous mutations/polymorphisms of the POR gene, encoding NADPH:cytochrome P450 oxidoreductase (CYPOR), have been described in patients with Antley-Bixler syndrome (ABS), presenting with craniofacial dysmorphogenesis, and/or disordered steroidogenesis, exhibiting ambiguous genitalia. CYPOR is the obligate electron donor to 51 microsomal cytochromes P450 that catalyze critical steroidogenic and xenobiotic reactions, and to two heme oxygenase isoforms, among other redox partners. To address the molecular basis of CYPOR dysfunction in ABS patients, the soluble catalytic domain of human CYPOR was bacterially expressed. WT enzyme was green, due to air-stable FMN semiquinone (blue) and oxidized FAD (yellow). The ABS mutant V492E was blue-gray. Flavin analysis indicated that WT had a protein:FAD:FMN ratio of approximately 1:1:1, whereas approximately 1:0.1:0.9 was observed for V492E, which retained 9% of the WT k(cat)/K(m) in NADPH:cytochrome c reductase assays. V492E was reconstituted upon addition of FAD, post-purification, as shown by flavin analysis, activity assay, and near UV-visible CD. Both Y459H and V492E were expressed as membrane anchor-containing proteins, which also exhibited FAD deficiency. CYP4A4-catalyzed omega-hydroxylation of prostaglandin E1 was supported by WT CYPOR but not by either of the ABS mutants. Hydroxylation activity was rescued for both Y459H and V492E upon addition of FAD to the reaction. Based on these findings, decreased FAD-binding affinity is proposed as the basis of the observed loss of CYPOR function in the Y459H and V492E POR mutations in ABS.


Assuntos
Craniossinostoses/genética , Flavina-Adenina Dinucleotídeo/química , Mutação , NADPH-Ferri-Hemoproteína Redutase/genética , Catálise , Dicroísmo Circular , Clonagem Molecular , Flavinas/química , Humanos , Cinética , Modelos Moleculares , Mutagênese , NADPH-Ferri-Hemoproteína Redutase/fisiologia , Oxirredução , Ligação Proteica , Síndrome
12.
Biochem Biophys Res Commun ; 338(1): 543-9, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16154533

RESUMO

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.


Assuntos
Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nitroso/química , Óxido Nitroso/metabolismo , Animais , Escherichia coli , Cromatografia Gasosa-Espectrometria de Massas/métodos
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